Carbonic anhydrase

Analytical gel filtration was performed using HR 10/30 column packed with Superdex 200 pg matrix attached to an ÄKTA Basic HPLC system. The column was equilibrated with a buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM KCl and calibrated with the standards β-amylase, alcohol dehydrogenase, bovine serum albumin, carbonic anhydrase and cytochrome c (Sigma-Aldrich). A sample volume of 100 μl containing 20 μM of PfGATase and PfATPPase was injected into the column individually and eluted at a flow rate of 0.5 ml min⁻¹ and the eluted proteins were detected at 220 nm. Similar conditions were used for PfGMPS and its mutants.

Purified PL3 samples were analyzed by MALDI-TOF in a Voyager DEPRO (Applied Biosystems), as described elsewhere (Moreno et al., 2008 (link)). A grid voltage of 89%, a 0.25 ion guide wire voltage, and a delay time of 400 ns in the linear positive-ion mode were used. External calibration was performed with carbonic anhydrase (29024 Da) and enolase (46672 Da) from Sigma, covering an m/z range of 10000–80000 units.

Gel filtration was performed on the XK 16/100 Superdex 200 column (GE Healthcare) balanced with 0.02 M Tris–HCl (pH 7.0) containing 0.5 M NaCl. The following protein markers (Sigma-Aldrich) were used to build the calibration curve: carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa). GDHs with the cut-off His-tag were used to determine molecular weights of the enzymes.

The pattern of protein hydrolysis and molecular weight of enzyme-treated chicken foot collagen were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC), respectively. To analyze the pattern of protein hydrolysis, we used the SDS-PAGE protocol described by Laemmli [21 (link)] with 12% separating and 5% stacking gels. Samples were prepared for electrophoresis by boiling in a loading buffer containing 0.1% Coomassie Brilliant Blue R 250 dye (Sigma-Aldrich, USA). Molecular weight distribution and average molar mass were evaluated using an LC-2000 Plus HPLC system (Jasco, Japan). For evaluation, the collagen samples were prepared by filtration through a 0.45-μm membrane using a Shodex Protein KW-802.5 column (I.D. 8 mm × 300 mm; Shodex, Japan). The mobile phase used was 50 mM phosphate buffer containing 0.3 M NaCl at a flow rate of 0.9 ml/min, and the detector recorded absorbance at 220 nm. We used thyroglobulin (669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa), aprotinin (6.5 kDa), and cyanocobalamin (1.3 kDa) as protein standards (all obtained from Sigma-Aldrich) for standard curve construction and evaluation of the average molecular weight of enzyme-hydrolyzed collagen samples.